There is a desperate need for new antibiotics to treat bacterial infections. The goal of this proposal is to develop a High-Throughput Screening (HTS) approach to identify small molecule inhibitors of bacterial Transcription Factors (TFs) that will serve as lead compounds in the development of new antibiotics. There are many known examples of TFs that are required for the virulence of pathogenic bacterial species. Furthermore, the mechanism of action of most bacterial TFs is extremely well understood. Hence, TFs represent excellent targets for antibacterial therapy. We have selected two TFs, cyclic-AMP receptor protein (CRP) and SoxS, which are required for the virulence and/or antibiotic resistance of multiple bacterial species. We hypothesize that chemical inhibitors of CRP or SoxS will be effective antimicrobials that inhibit virulence or counteract antibiotic resistance of multiple important pathogens; no such inhibitors are currently available. Furthermore, the HTS approach we develop will serve as a generic system for identifying inhibitors of any bacterial TF. We will develop HTS assays that give reproducible, robust signal with low noise. These will be based on previously-developed proof-of-principle assays, including a bioluminescence assay for CRP function that gives a high Z' score (>0.86 in 96-well format). The independent HTS assays for CRP and SoxS will serve as controls for each other, eliminating the need for secondary screens. We will test these assays by performing screens with small libraries of compounds. This will establish the applicability of the screens to HTS, and will determine their suitability for miniaturization. We will validate and characterize putative CR and SoxS inhibitors using a battery of well-established in vivo and in vitro assays. We expect to develop robust, highly reproducible assays for CRP and SoxS function that can be applied to HTS. Once validated, the assays will be submitted to the Molecular Libraries Screening Centers Network (MLSCN) for HTS. The CRP and SoxS inhibitors we expect to identify in this work and in future HTS screening represent lead compounds in the development of novel antimicrobials. In future work we will test the efficacy of these compounds using in vitro and in vivo infection models for Salmonella Typhimurium, a bacterium responsible for millions of infections annually in the US, and Yersinia pestis (plague), a major bioterrorism threat.